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Dyes

Dyes. Photostimulation: Channel Rhodopsin Caged glutamate (and others) Ca 2+ sensitive Voltage-Sensitive dyes FRET, GRASP Quantum Dots Ultra-High-Resolution Light Microscopy. Channel Rhodopsin/Halorhodopsin. Channel Rhodopsin - structure. Many thanks to Brikha !.

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Dyes

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  1. Dyes • Photostimulation: • Channel Rhodopsin • Caged glutamate (and others) • Ca2+ sensitive • Voltage-Sensitive dyes • FRET, GRASP • Quantum Dots • Ultra-High-Resolution Light Microscopy

  2. Channel Rhodopsin/Halorhodopsin

  3. Channel Rhodopsin - structure Many thanks to Brikha! Gary G. Matthews, Neurobiology book

  4. Channel Rhodopsin Switch

  5. ChR2, NpHR pros/cons • Pros Substitution for stimulating electrodes Stimulation of a great many cells at once Genetic targeting of cells subsets Less invasive compare to electrophysiology • Cons Hard to get expression in a single cell Hard to get to the deep tissue (fiber-optics) Variable expression levels, hence unknown response

  6. Glutamate uncaging • Release glutamate upon illumination with 355nm (ultraviolet) light • Temporal spike jitter: 1-3ns (10ns) • Spatial resolution: ~5μm • Other caged compounds: GABA, glycine • ROPING (Reverse Optical Probing

  7. Ca2+ imaging • Calcium indicators • Good SNR • Saturation • Slow Signal (decay ~2s) • New statistical techniques for optical inference Paul De Koninck, 2007 !! VIDEO DEMO!! Vogelstein et al, 2009

  8. Voltage-sensitive dyes • Usually spans the membrane • Dipoles orient themselves and change value ~ΔV • For one cell in vertebrates : Low SNR • But works great in Invertebrates! (Briggman, Kristan – leech, now – tritonia (>100 neurons, <1ms resolut.) www.umsl.edu/~tsytsarev/tsytsarev_files/Lecture10.htm

  9. FRET (Förster Resonance Energy Transfer) sorry, no references for the figures

  10. FRET (Förster Resonance Energy Transfer) • Is extensively used in protein-protein interaction studies • Occurs only when proteins get closer than 10nm • Energy is transferred via induced dipoles of Fluorophores • Formally the emission-absorption spectra can be used • Carl Schoonover NSF proposal • GRASP (GFP Reconstitution Across Synaptic Partners) - Evan H. Feinberg (Cori Bargmann)

  11. Quantum Dots • Nanoscale semiconductors • The emission frequency depends on the particle size (the larger, the redder) • Problem is how to deliver inside

  12. Ultra-high-resolution light microscopy • STED (Stimulated Emissions Depletion) / GSD (Ground State Depletion) - RESOLFT • PALM (Photo-Activated Localization Microscopy) / FPALM (Fluorescent PALM) / STORM (STochastic Optical Reconstruction Microscopy) • SI (Structures Illumination) (wide-field)

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